Engineering of Thermostable β-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines

Article abstract of DOI:10.1002/cbic.202000526

The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-βHAD), which catalyzes the NADP⁺-dependent oxidation of β-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-βHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.

Full text of DOI:10.1002/cbic.202000526

Communications
doi.org/10.1002/cbic.202000526
ChemBioChem
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Engineering of Thermostable β-Hydroxyacid
Dehydrogenase for the Asymmetric Reduction of Imines
Peter Stockinger+,[a] Luca Schelle+,[a] Benedikt Schober,[a] Patrick C. F. Buchholz,[a]
Jürgen Pleiss,[a] and Bettina M. Nestl*[a]
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Imine Reductase Engineering Database (https://ired.biocat-
net.de/).[30,31] A deeper analysis of the sequence space of IRED
homologues revealed a significant sequence similarity and a
similar quaternary structure of β-hydroxyacid dehydrogenases
(βHADs).[32] In addition to IREDs, several short-chain dehydro-
genases (SDRs) were reported to catalyze imine reduction.[33,34]
In contrast to βHADs, SDRs lack the sequence and structural
similarities to IREDs and βHADs and constitute a separate, large
enzyme family (https://sdred.biocatnet.de/).[35] By systematically
comparing imine-reducing representatives of IREDs, βHADs, and
SDRs, common principles were derived to support the targeted
engineering of SDRs and βHADs into imine-reducing enzymes
(Scheme 1).[36] The existence of alternative proton donors in
IREDs and βHADs was proposed, and functional-relevant
residues were identified. These flank the active site and mediate
the local electrostatic fine-tuning of the catalytic residues.[36]
Exchanging several flanking residues in a dehydrogenase with
distinct carbonyl-reducing activity resulted in a promiscuous
enzyme with imine-reducing activity.[37] Recently, three new
imine-reducing enzymes were generated by introducing single-
point mutations into βHADs, including the exchange of the
proton donor and the removal of a bulky gate-keeping
residue.[38] To access imine reduction in the glyoxylate reductase
from Arabidopsis thaliana (At-βHAD), the catalytically essential
lysine at position 170 (K170) was exchanged by aspartic acid.
The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-
βHAD), which catalyzes the NADP+-dependent oxidation of β-
hydroxyacids, was engineered to accept imines as substrates.
The catalytic activity of the proton-donor variant K189D was
further increased by the introduction of two nonpolar flanking
residues (N192 L, N193 L). Engineering the putative alternative
proton donor (D258S) and the gate-keeping residue (F250 A)
led to a switched substrate specificity as compared to the single
and triple variants. The two most active Ta-βHAD variants were
applied to biocatalytic asymmetric reductions of imines at
elevated temperatures and enabled enhanced product forma-
°
tion at a reaction temperature of 50 C.
The asymmetric reduction of imines to allow the formation of
chiral primary, secondary, and tertiary amines has been
identified as a key area in synthetic organic chemistry.[1] In this
context, the development of enzymatic strategies is highly
attractive because of their mild reaction conditions and their
excellent selectivity (chemo-, regio- and stereoselectivity).
Successful biocatalysts have been reported for the synthesis of
chiral amines from different enzyme classes, including lipases,
monoamine oxidases, ω-transaminases, amine dehydrogenases,
ammonia lyases, engineered cytochrome P411, and imine
reductases.[2–8] In particular, imine reductases (IREDs) have
emerged as a valuable new set of biocatalysts for the
asymmetric synthesis of optically active amines.[1,9–17] IREDs and
related reductive aminases have been applied in biotransforma-
tions and enzyme cascades for the synthesis of chiral
amines.[18–29]
The use of sequence-based bioinformatics classification
approaches enabled the identification of characteristic se-
quence motifs and the annotation of putative IREDs in the
[a] P. Stockinger,+ L. Schelle,+ B. Schober, Dr. P. C. F. Buchholz, Prof. Dr. J. Pleiss,
Dr. B. M. Nestl
Institute of Biochemistry and Technical Biochemistry
Department of Technical Biochemistry
University of Stuttgart, Germany
Allmandring 31, 70569 Stuttgart (Germany)
E-mail: bettina.nestl@itb.uni-stuttgart.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202000526
© 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
Scheme 1. Natural asymmetric reduction of ketoacids by β-hydroxyacid
dehydrogenases and novel imine-reducing activity by rational enzyme
engineering.
ChemBioChem 2020, 21, 1–5
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Notably, imine reduction was also observed upon mutation
information to elucidate beneficial mutations and using multi-
ple sequence alignments of enzyme homologues. In addition to
single mutation K189D in Ta-βHAD affording activity towards 3,
further positions were considered to play an important role in
catalytic activity and even altering substrate specificity.[38] Given
the previous observation that the K170D/N174L/D239A muta-
tion in At-βHAD rendered the instable K140D/N174L variants
active (https://doi.org/10.18419/opus-10763), residues N192 and
N193 in Ta-βHAD were selected as positions to introduce
potentially beneficial nonpolar flanking residues. Moreover,
residues D258 and F250, considered as a putative alternative
proton donor and gate-keeping residue, respectively, were
included to perform combinatorial single-site mutagenesis
(Table S5). Figure 1 highlights residues (i.e., N192, N193, F250,
and D258) that are proposed to be important for imine
reduction in Ta-βHAD.
Considering these positions, an intuitively, focused combi-
natorial library was designed to further minimize the screening
effort. In total, 20 variants were generated using Ta-βHAD
K189D as a template. These engineered and purified variants of
Ta-βHAD were tested with a panel of five imine substrates
(Figure 2, Tables 1 and S8).
Biotransformations were analyzed by achiral and chiral GC
(substrates 1 and 3) and HPLC (substrates 2a–2c). No activity
was observed with substrate 1 as well as dihydroisoquinoline
substrates 2b and 2c, possibly as a result of more sterically
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of K170 to phenylalanine. The presence of the polar residues
asparagine (N174) and aspartic acid (D240) might rationalize
the observed imine-reducing activity. This initial report estab-
lished a proof-of-concept and highlighted limitations, notably
low stability and expression level of the generated βHAD
variants. At-βHAD double-variant K170D/N174L resulted in an
unstable enzyme. Given the lack of polar interactions with
N174, unfavorable repulsive energies between the neighboring
aspartic acid residues are conceivable. Indeed, the destabilizing
effect of the double variant K170D/N174 was compensated in
the triple variant K170D/N174L/D239A and resulted in a
catalytically active protein (https://doi.org/10.18419/opus-
10763).
We thus envisaged developing thermostable imine-reduc-
ing enzymes for further application in the synthesis of chiral
amines. As IREDs from thermophilic organisms are rare, the
larger family of βHADs served as a starting point for thermo-
stable-enzyme candidates. Several βHAD homologues from
hyperthermophiles were identified by comparison with the
BacDive database.[39] Four candidates were selected for further
analysis (details of the sequence selection are given in the
Supporting Information): Tt-βHAD-1 and Tt-βHAD-2 from Ther-
mus thermophilus, Ta-βHAD from Thermocrinis albus and Pc-
βHAD from Pyrobaculum calidifontis with pairwise protein
sequence identities ranging from 27 to 36% (see Tables S1 and
S2 in the Supporting Information). As described previously,[1],
the four selected genes were synthesized carrying the lysine to
aspartic acid mutation and subcloned into a pBAD-33 vector
followed by overexpression in Escherichia coli JW5510. After
heat-shock purification, the thermofluor assay[40] with Sypro
Orange was used to determine the thermostability. The thermal
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°
denaturation temperature of selected βHADs was about 60 C
(Table 1, Figures S6–S9).
Further, the oligomerization state was analyzed by size-
exclusion chromatography. While the Pc-βHAD K190D variant
was observed to form a dimer, Tt-βHAD-1, Tt-βHAD-2, and Ta-
βHAD variants were found to oligomerize as tetramers
(Figures S2-S5). To determine whether these single βHAD
variants possessed activity towards imines, substrates 2-meth-
ylpyrroline (1) and 3,4-dihydroisochinoline (2a) were first
examined. None of the variants showed activity towards
substrate 1. Furthermore, we also evaluated the reduction of 1
using wild-type enzymes. No promiscuous imine reduction
activity was found. However, good product formation (60.5�
1.0%) was observed with imine 2a and variant Ta-βHAD K189D
°
at 25 C and 24 h biotransformation time.
Engineering the active site of βHADs was effective to alter
the catalytic activity and substrate specificity by using structural
Table 1. Thermostabilities (Tampp) of variants.
Apparent melting temperature (Tmapp
)
Variant
Figure 1. Imine-reducing active-site variants resulting from combinatorial
mutagenesis of Ta-βHAD visualized in a homology model. A) Single variant
TA1 (K189D). B) Triple variant TA6 (K189D/N192L/N193L). C) Quadruple
variant TA15 (K189D/N192L/N193L/F250A). D) Quintuple variant TA20
(K189D/N192L/N193L/F250A/D258S).
°
Tt-βHAD-1K185D
Tt-βHAD-2K187D
Ta-βHAD K189D
Pc-βHAD K190D
61.8�0.2 C
°
56.9�0.8 C
°
60.5�0.4 C
°
59.8�0.8 C
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K189D/N192L/N193L/F25A/D258S) demonstrated the highest
1
2
3
product formation. Product formation of 77% and a high
enantiomeric excess 99% for the S-product was detected after
24 hours of biotransformation (Figures S11 and S12).
4
The increased product formation for variant T4 supports the
need for a nonpolar donor-flanking residue. Variant T6 (Fig-
ure 1B) displays an additively increased product formation,
which could also be rationalized by the need for a nonpolar
environment of the proton donor, but also by an advantageous
influence on the binding of the nonpolar substrates. Variant
TA15 was the only variant that displayed productive conver-
sions of both substrate 2a and 3. As the only difference
compared to variant T6 was introduced by the additional
mutation F250A, an erosion of steric effects negatively influenc-
ing the binding of substrate 3 is assumed. The drop of activity
observed for substrate 2a might be rationalized by the direct
involvement of F250 in productive binding. Therefore, the term
gate-keeper does only apply for the imine-reducing activity
toward substrate 3. Interestingly, the product formation with
substrate 2a is lost with the additional mutations D258A or S in
variants T17–T20, which supports the hypothesis of alternative
proton donors in βHADs.[1] However, it cannot be excluded that
the altered steric and electrostatic properties of this additional
mutation hinder the productive binding of substrate 2a .
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Figure 2. Imine substrates used in the asymmetric reduction with newly
engineered Ta-βHAD.
demanding methyl and methoxy substituents. Interestingly,
two variants (TA4 and TA6) were found to exhibit improved
activity for substrates 2a and 3. For example, triple variant TA6
(Ta-βHAD K189D/N192 L/N193L) showed high activity (>99%
product formation) with 2a after 4 h of biotransformation
(Table 2, Figure S10). Remarkably, variant TA15 (Ta-βHAD
K189D/N192L/N193L/F250A) displayed activity on both sub-
strates 2a and 3. Improved conversions were obtained for 3
with various variants, whereas quintuple variant TA20 (Ta-βHAD
Finally, to demonstrate the thermostable imine-reducing
applicability of variants generated, the asymmetric reductions
of imines 2a and 3 were performed at elevated temperatures.
°
The reactions were compared for temperatures 25 and 50 C
using variants TA6 and TA20 in the reductions of substrates 2a
and 3, respectively. As a full conversion of 2a was observed
°
with TA6 at 25 C within 4 h reaction time, the amount of
enzyme was reduced to monitor the impact of temperature on
enzyme activity. Thus, the enzyme concentrations were lowered
(TA6 1 mgmLÀ 1 and TA20 2.5 mgmLÀ 1) to avoid full product
formation (Table 3). Improved asymmetric reduction of 2a and
Table 2. Activities of Ta-βHAD variants towards 3,4-dihydroisoquinoline
(2a) and 2-phenylpiperideine (3) after 24 h reaction time.
Variant
Product formation [%] with
1, 2b and 2c
°
2a[a]
3[a]
3 at 50 C was achieved with 50 and 30% product formations,
respectively. These results show an increase of 27% product
formation with TA4 (imine 2a) and 85% with TA 20 (imine 3),
while selectivity remained unchanged (99% ee for S-amine
product). To monitor the initial reduction activities towards
imines 2a and 3 at elevated temperatures, lower enzyme
concentrations were used.
TA1
TA2
TA3
TA4
TA5
TA6
TA7
TA8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
67.1�6.7
–
96.3�4.8
–
–
–
–
–
–
–
–
TA9
TA10
TA11
TA12
TA13
TA14
TA15
TA16
TA17
TA18
TA19
TA20
Table 3. Comparison of reductions of imines 2a and
3 at elevated
temperatures using the two best variants after 4 h reaction times.
Variant
Substrate[a]
T [ C]
Amine product [%][b]
ee [%][b]
32.0�0.1
43.9�4.0
°
–
–
–
–
–
–
13.1�0.7
13.9�0.2
44.9�1.0
76.6�2.0
1
2
3
4
TA6
TA6
TA20
TA20
2a
2a
3
25
55
25
55
38.7�0.6
49.5�2.8
16.1�1.2
29.6�1.4
n.a.
n.a.
99 (S)
99 (S)
3
[a] Reactions (150 μL) performed in Tris·HCl buffer (50 mM, pH 8) with
substrates (5 mM), NADPH (2.5 mM), glucose-6-phosphate (25 mM),
glucose-6-phosphate dehydrogenase (1 U mLÀ 1), MgCl2 (2.5 mM), and Ta-
βHAD variants (5 mgmLÀ 1). b Product formations were determined by HPLC
and GC analysis; see the Supporting Information. n.a.: not applicable. À : no
activity
[a] Reactions (150 μL) performed in Tris·HCl buffer (50 mM, pH 8) with
substrates (5 mM), NADPH (2.5 mM), glucose-6-phosphate (25 mM),
glucose-6-phosphate dehydrogenase (1 UmLÀ 1), MgCl2 (2.5 mM), and Ta-
βHAD variants TA6 (1 mgmLÀ 1) and TA20 (2.5 mgmLÀ 1). [b] Product
formations and ee values were determined by HPLC and GC analysis; see
the Supporting Information. n.a.: not applicable
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Acknowledgments
The research leading to these results has received support from
the German Research Foundation (Deutsche Forschungsgemein-
schaft, DFG, project number 284111627). Open access funding
enabled and organized by Projekt DEAL.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: beta-HAD
· imine reductases · promiscuity ·
substrate specificity · thermostability
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Biochem. 2006, 357, 289–298.
Manuscript received: July 31, 2020
Version of record online: ■■■, ■■■■
ChemBioChem 2020, 21, 1–5
www.chembiochem.org
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© 2020 The Authors. Published by Wiley-VCH GmbH
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COMMUNICATIONS
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Get more from your enzyme: A β-
hydroxyacid dehydrogenase from
Thermocrinus albus was engineered
to accept two different imine
compounds as substrates. The
variants were applied to biocatalytic
asymmetric reductions at elevated
temperatures leading to an
P. Stockinger, L. Schelle, B. Schober,
Dr. P. C. F. Buchholz, Prof. Dr. J. Pleiss,
Dr. B. M. Nestl*
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Engineering of Thermostable β-Hy-
droxyacid Dehydrogenase for the
Asymmetric Reduction of Imines
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enhanced product formation.